Goran Bačić

In Vivo Morphological Changes in Animal Models of Amyotrophic Lateral Sclerosis and Alzheimer's‐Like Disease: MRI Approach

Magnetic resonance imaging (MRI) is the only noninvasive technique that provides structural information on both cell loss and metabolic changes. After reviewing all the results obtained in clinical studies, reliable biomarkers in neurological diseases are still lacking. Diffusional MRI, MR spectroscopy, and the assessment of regional atrophy are promising approaches, but they cannot be simultaneously used on a single patient. Thus, for further research progress, reliable animal models are needed. To this aim, we have used the clinical MRI to assess neurodegenerative processes in the hSOD‐1G93A ALS rat model and in the trimethyltin (TMT)‐treated model of Alzheimers‐like disease. T2‐weighted (T2W) hyperintensive neurodegenerative foci were found in the brainstem of the ALS rat with apparent lateral ventricle dilation (T1W—hypointensity vs. T2W—hyperintensity). Degenerative processes in these areas were also confirmed by confocal images of GFAP‐positive astrogliosis. MRI after i.v.i. of magnetic anti‐CD4 antibodies indicated an accumulation of inflammatory cells near dilated ventricles. TMT‐treated rats also revealed the dilation of lateral ventricles. Expected deterioration in the hippocampus was not observed by clinical MRI, but immunocytochemistry could reveal significant redistribution of macro‐ and microglia in this structure. In both models, Gd‐DTPA contrast revealed a compromised blood brain barrier that may serve as the passage for inflammatory immune cells in the vicinity of dilated lateral ventricles. Moreover, in both models the midbrain region of the dorsal hippocampus was the target of BBB compromise, thus revealing a potentially vulnerable point that can be the primary target of neurodegeneration in the central nervous system. Anat Rec, 292:1882–1892, 2009.

Brain iron MRI: A biomarker for amyotrophic lateral sclerosis

To evaluate the usefulness of MRI detection of hypointensity areas (iron deposits) in the brain using a dedicated MRI technique in patients with ALS in establishing this sign as a potential surrogate biomarker that correlates with the severity of disease.

Detection of diffusion and distribution of oxygen by fast-scan EPR imaging

Abstract The diffusion and distribution of oxygen in models of biological systems have been followed by a fast-scan 2D EPR imaging apparatus. The EPR imager is based on the use of two sets of gradient coils with computer control to select projections. Currently the time per projection (six seconds) is sufficient to follow oxygen diffusion in the model systems. Typically 18–20 projections are obtained to generate an image. Using physical rotation of x/y coils, this apparatus can be used for three-dimensional EPR imaging.

Effects of 5-fluorouracil on erythrocytes in relation to its cardiotoxicity : Membrane structure and functioning

In the present study, we showed that the antineoplastic drug 5-fluorouracil (5-FU) induces in vitro exposure-time/dose-dependent changes on the level of an erythrocytes morphology, ionic balance, and membrane fluidity. These changes are partially or fully irreversible, and we suggest that they are provoked by an irreversible depletion of ATP. Because of these changes that could also occur in vivo during 5-FU infusion, a certain amount of erythrocytes with echinocytic shape and diminished ability to deliver oxygen is present in blood for longer periods of time. This renders oxygen transport and delivery more difficult, leaving the heart with an insufficient supply of oxygen, thus leading to cardiotoxicity.

EPR Spin Trapping of Oxygen Radicals in Plants: A Methodological Overview

Abstract: We present a brief account of the difficulties involved in detection of oxygen free radicals in plants and give a rationale for using the EPR spin trapping technique in such studies. Comparative analysis of characteristics of different spin traps is given, having in mind their suitability in trapping oxygen‐centered free radicals. Certain technical aspects of EPR experiments related to successful trapping of free radicals are discussed. Previous studies of trapping of oxygen radicals in plants are reviewed in terms of how efficient the experimental approach employed has been in their detection and how this influences conclusions about the mechanisms of their production. In addition, we analyze the potential of spin labels in the analysis of free radical production in plants and demonstrate that the combination of EPR spin traps and spin labels is extremely efficient for this purpose.

Imaging cellular markers of neuroinflammation in the brain of the rat model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disorder affecting upper and lower motoneurons. Since immune disbalance is known to be an important manifestation of the disease, working with the familial ALS rat model, hSODG93A (containing multiple copies of the human SOD1 G93A mutation), we were particularly interested in following by live magnetic resonance imaging (MRI) the immune cells labeled by ultra small paramagnetic iron oxide (USPIO) nanoparticles. In addition, microglial activation was studied by immunocytochemistry. MRI of USPIO labeled Tcells revealed CD4+ lymphocyte infiltration in the midbraininterbrain region while the CD8+ cells were more confined to the brainstem region. By way of gadolinium (Gd) contrast it was also confirmed that the bloodbrain barrier (BBB) was compromised. Moreover, it was revealed that the regions of BBB breakthrough were congruent with the MRI foci of Tcell infiltration. Immunocytochemistry revealed microglial activation and fusion, possibly phagocytic interactions with neurons in the hippocampus and brainstem. These observations prove the existence of an elaborate inflammatory process in the brain of hSODG93A rats, and also demonstrates the complexity and multifocality of ALS as having its inflammatory manifestations also in the central nervous system (hippocampus) distinct from clinically described motor foci of degeneration.

Detection of Oxygen-Centered Radicals Using EPR Spin-Trap DEPMPO: The Effect of Oxygen

Abstract: Studies of the ability of the EPR spin trap DEPMPO to detect both superoxide and hydroxyl radicals produced by systems in vitro and in vivo are presented. Experiments using free radical‐generating systems confirmed the suitability of the EPR spin trapping technique but also revealed the existence of an undesirable conversion of DEPMPO/OOH into DEPMPO/OH adducts. The rate of conversion decreases with oxygenation, and the production of oxygen‐centered radicals increases. However, this property of DEPMPO does not have a significant influence on its ability to independently detect radicals produced by plant plasma membranes. Since the adduct conversion appears to be rather slow compared to radical generation, we conclude that the DEPMPO spin‐trap can be efficiently used for detection of oxygen‐centered radicals produced by systems in vivo, as demonstrated for isolated plasma membranes.

In vivo evaluation of different alterations of redox status by studying pharmacokinetics of nitroxides using magnetic resonance techniques

Free radicals, particularly reactive oxygen species (ROS), are involved in various pathologies, injuries related to radiation, ischemia-reperfusion or ageing. Unfortunately, it is virtually impossible to directly detect free radicals in vivo, but the redox status of the whole organism or particular organ can be studied in vivo by using magnetic resonance techniques (EPR and MRI) and paramagnetic stable free radicals – nitroxides. Here we review results obtained in vivo following the pharmacokinetics of nitroxides on experimental animals (and a few in humans) under various conditions. The focus was on conditions where the redox status has been altered by induced diseases or harmful agents, clearly demonstrating that various EPR/MRI/nitroxide combinations can reliably detect metabolically induced changes in the redox status of organs. These findings can improve our understanding of oxidative stress and provide a basis for studying the effectiveness of interventions aimed to modulate oxidative stress. Also, we anticipate that the in vivo EPR/MRI approach in studying the redox status can play a vital role in the clinical management of various pathologies in the years to come providing the development of adequate equipment and probes.

Decreased oxygen transfer capacity of erythrocytes as a cause of 5-fluorouracil related ischemia.

Various mechanisms have been proposed to account for chemotherapy related ischemia, but none of them can explain the available clinical data. In order to explore the possibility that the decreased ability of erythrocytes to deliver oxygen to the heart could be responsible for cardiotoxicity, we have performed an ex vivo and in vivo study of the effects of cisplatin/5-FU on erythrocytes, using a variety of biophysical techniques. Combining EPR and microscopy it was concluded that both cardiotoxic 5-FU and non-cardiotoxic cisplatin have similar effects on the erythrocyte membrane, thus eliminating those changes as a potential source of cardiotoxicity. On the contrary, 31P-NMR and polarography showed that the effects of these cytostatics on the intracellular milieu differ significantly. 5-FU provoked a pronounced decrease of the O2 level in blood and affected the metabolism of phosphate compounds, while cisplatin had no such effects. When combined these two drugs showed synergistic effects, which matches the higher frequency of cardiotoxicity of the combination relative to the sole application of 5-FU. Preliminary results acquired on blood of patients receiving cisplatin/5-FU therapy verified observations obtained ex vivo. These results open a possibility of applying NMR in preclinical trials of new drugs in order to predict their ischemic potential.

31P NMR Spectroscopy and Polarographic Combined Study of Erythrocytes Treated with 5‐Fluorouracil: Cardiotoxicity‐Related Changes in ATP, 2,3‐BPG, and O2 Metabolism

Abstract: Antineoplastic drug 5‐fluorouracil (5‐FU) frequently shows cardiotoxic effects, the mechanism of which has not yet been elucidated. The objective of the present study was to explore effects of 5‐FU on metabolism of ATP, 2,3‐BPG, and oxygen in erythrocytes and to relate these to the phenomenon of 5‐FU cardiotoxicity. We determined that 5‐FU induced rapid increase in O2 consumption, which led to drastic changes in the metabolism of phosphate compounds in erythrocytes. Decrease in pO2 provoked increase in production of 2,3‐BPG and subsequent deoxygenation of oxyHb to deoxyHb. However, the most important effect of 5‐FU on erythrocytes is severe decrease in the level of ATP. This could lead to a number of irreversible changes in erythrocyte structure and functioning, such as echinocytosis, increase in membrane fluidity, and non‐functioning of membrane ion pumps. All these changes affect normal functioning of erythrocytes, leading to difficulties in oxygen transport and insufficient supply of oxygen to the heart, and pointing to the importance of studying the effects of antineoplastic drugs on intracellular metabolism of erythrocytes.